US7476545B2 - Method of quickly detecting and/or assaying antigen by fluorescence correlation spectrometry - Google Patents

Method of quickly detecting and/or assaying antigen by fluorescence correlation spectrometry Download PDF

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US7476545B2
US7476545B2 US11/633,345 US63334506A US7476545B2 US 7476545 B2 US7476545 B2 US 7476545B2 US 63334506 A US63334506 A US 63334506A US 7476545 B2 US7476545 B2 US 7476545B2
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antigen
fluorescence
labeled
antibody
correlation spectroscopy
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US20070154950A1 (en
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Masataka Kinjo
Motohiro Horiuchi
Fumihiko Fujii
Hiroshi Sakata
Mamoru Tamura
Masayoshi Ueno
Takayuki Yanagiya
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Japan Science and Technology Agency
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases

Definitions

  • the present invention relates to a method of quickly detecting and/or assaying an antigen, whereby an antigen such as an antigenic protein, for example, a pathogenic protein (e.g., an abnormal prion) or a harmful protein contained in a food material is detected and/or assayed quickly by using fluorescence correlation spectroscopy.
  • an antigen such as an antigenic protein, for example, a pathogenic protein (e.g., an abnormal prion) or a harmful protein contained in a food material is detected and/or assayed quickly by using fluorescence correlation spectroscopy.
  • harmful proteins include an allergen protein contained in food materials such as buckwheat, wheat, and rice.
  • pathogenic protein includes an abnormal prion (infectious) contained in materials for edible meat and meat-and-bone meal.
  • an abnormal prion is a protein that causes prion disease typified by bovine spongiform encephalopathy (BSE).
  • BSE bovine spongiform encephalopathy
  • a normal prion protein is a glyocoprotein that is commonly present in animal brain and on the neural cell membrane surface, and has a molecular weight of approximately thirty-three thousand to thirty-five thousand Daltons(33 to 35 kDa).
  • its infectious prion protein form is intracellularly accumulated in the brain (Lait, 76: 571-578, 1996).
  • abnormal prions convert normal prions, which are produced at particular sites in the body, into abnormal prions. This results in the accumulation of the abnormal prions at those particular sites. The accumulation of the abnormal prions in the brain renders the brain spongiform, leading to animal death.
  • Immunoassay such as ELISA (enzyme-linked immunosorbent assay) or Western blotting (immunoblotting) has here to fore been used to assay natural biological proteins such as prions (abnormal).
  • ELISA is a method performed on a solid phase, wherein an antigen or an antibody is labeled with an enzyme, and the presence of the antibody or the antigen is detected by use of the enzyme activity.
  • ELISA can involve a procedure of binding a Mab 3F4 antibody to a prion immobilized on a microtiter plate and detecting this antibody with a second antibody; the second antibody is coupled to an enzyme which catalyzes a coloring reaction that can be detected (U.S. Pat. No. 4,806,627).
  • Western blotting is a method wherein a protein separated by electrophoresis is immobilized on a hydrophobic membrane, and the protein of interest is detected with an antigen-specific antibody.
  • the detection of an abnormal prion by Western blotting is performed, for instance, by a procedure using a monoclonal anti-prion protein antibody Mab 13A5 (J. Infect. Dis. 154: 518-521, 1986).
  • the method involves performing in advance, for example, a procedure of digestion and removal of a normal prion from a test sample by treatment with proteinase K. This procedure is for detecting an abnormal prion separately from a normal prion.
  • Western blotting also requires performing electrophoresis. These methods involve complexities and takes much time.
  • ELISA requires subjecting a sample to denaturation treatment with guanidine thiocyanate after proteinase K treatment.
  • ELISA also requires performing primary denaturation treatment with SDS and a protein concentration procedure by methanol treatment before the deaggregation of the prion protein.
  • centrifugation must be performed both before the methanol treatment and before the treatment with guanidine thiocyanate. This centrifugation procedure can take much time. In all, these methods involve complicated treatments and therefore present a problem of being unsuitable for testing a large number of samples in a short period of time.
  • Japanese Patent Application No. 10-267928 relates to an immuno-PCR method that applies ELISA in detecting an abnormal prion protein with high sensitivity, wherein an anti-prion protein antibody is used and labeled with an arbitrary DNA fragment that is detected by PCR.
  • Japanese Patent Application No. 2003-130880 relates to a method of immunoassaying an abnormal prion with high sensitivity without performing a time-consuming electrophoresis or centrifugation procedure of the conventional ELISA or Western blotting methods.
  • a first antibody for inducing an antigen/antibody reaction with an abnormal prion treated with a denaturing agent, or an antigen-binding fragment thereof is immobilized on magnetic particles and used as an immunoassay reagent.
  • the method assays the abnormal prion without performing the centrifugation procedure or electrophoresis, and can test a large number of samples in a short time.
  • Japanese Patent Application No. 2003-215131 relates to a method of analyzing a prion protein by using a mass spectrum, wherein a prion protein in a body fluid sample is allowed to form a covalent bond by reaction with a chemical. Hence, in the presence of a pathogenic prion, at least one additional peak is observed in the mass spectrum.
  • FCS fluorescence correlation spectroscopy
  • This method can detect and assay, in almost real time, the physical parameters of protein molecules such as number, size, or shape, without undergoing a step of physically separating the sample (Chem. Phys., 4, 390-401, 1974; Biopolymers, 13, 1-27, 1974; Physical Rev. A, 10: 1938-1945,1974; in Topics in Fluorescence Spectroscopy, 1, pp. 337-378, Plenum Press, New York and London, 1991; and R. Rigler, E. S. Elson (Eds.), Fluorescence Correlation Spectroscopy.
  • FCS is practiced by capturing, within an exceedingly small region, the Brownian motions of fluorescence-labeled target molecules in a medium by a laser confocal scanning microscope system. This provides an analysis of the diffusion time from the fluctuation of fluorescence intensity and an assay of the physical parameters of the target molecules (the number and size of the molecules). Thus, FCS serves as an effective means in specifically detecting intermolecular interaction with high sensitivity.
  • FCS used in the detection and assay of a protein, or the like, contained in a biological sample is that the concentrations or intermolecular interactions of fluorescence-labeled target molecules contained in a solution can be monitored in almost real time without undergoing a physical separation step. Therefore, a detection system using FCS can avoid a complicated Bound/Free separation step necessary for conventional analysis means (e.g., ELISA) and predominantly used in biomolecule detection systems. This technique can assay large amounts of samples with high sensitivity in a short time and is also suitable for automatic assay.
  • a fluorescence-labeled antibody molecule is used, and an antigen/antibody reaction occurs between the fluorescence-labeled antibody and the antigenic protein.
  • Analysis is performed by utilizing the difference in diffusion rate that occurs due to the shapes and molecular weights of the fluorescence-labeled antibody and the fluorescence-labeled antibody/antigenic protein complex.
  • the diffusion rate (diffusion constant or D) refers to an area where molecules are freely diffused per unit time.
  • the diffusion time (DT or ⁇ D) refers to time required for molecules to pass through a determined focal region that depends on the apparatus.
  • FCS provides an accurate assay of an antigenic protein or the like in a sample by using the difference in diffusion rates between the labeled antibody and the antigen/antibody complex formed by the labeled antibody and the antigenic protein.
  • FCS could previously detect only the exceedingly limited type of an antigenic protein or the like due to this requirement.
  • Conventional means for solving this problem comprised applying a variety of modifications to an antigen/antibody complex in consideration of the shapes and molecular weights of the antigen and the antibody and providing a significant difference in diffusion rate (Japanese Patent Application No. 2001-272404 and Japanese Patent No. 3517241). However, even if these methods were used, the applicability of FCS was limited regarding which objects could be detected.
  • An object of the present invention is to provide a quick and convenient method of detecting and/or assaying an antigen or an antigenic protein such as a pathogenic protein (e.g., an abnormal prion) or a harmful protein contained in a food material.
  • an antigen or an antigenic protein such as a pathogenic protein (e.g., an abnormal prion) or a harmful protein contained in a food material.
  • FCS fluorescence correlation spectroscopy
  • the present inventors discovered that, according to this method, a wide scope of antigens can be detected and assayed using FCS, including antigens that have relatively low molecular weight such as an antigenic protein, independent from the shape or molecular weight of the antigen. Based on these findings, the present invention has been completed.
  • the present invention may comprise a method of detecting and/or assaying an antigen, such as an antigenic protein, by using a fluorescence-labeled antibody fragment targeted to an epitope of the antigen and a non-fluorescence-labeled intact antibody targeted to another epitope of the antigen which together forms an antigen/antibody complex among the antigen, the fluorescence-labeled antibody fragment, and the non-fluorescence-labeled intact antibody.
  • the method may further comprise detecting and analyzing the formed antigen/antibody complex by fluorescence correlation spectroscopy.
  • the detection and/or assay method of the present invention can be applied widely to the detection and/or assay of an antigenic protein, for example, a pathogenic protein (e.g., an abnormal prion) or a harmful protein contained in a food material, whereby the antigenic protein can be detected and/or assayed quickly and accurately by a simple procedure.
  • an antigenic protein for example, a pathogenic protein (e.g., an abnormal prion) or a harmful protein contained in a food material, whereby the antigenic protein can be detected and/or assayed quickly and accurately by a simple procedure.
  • the present invention may comprise (i) mixing a fluorescence-labeled antibody fragment and an intact antibody (or a mixture thereof, i.e., an antigen detection reagent provided by the present invention) with an antigen such as an antigenic protein (e.g., a pathogenic protein such as an abnormal prion or a harmful protein such as an allergen protein), in which the antibody fragment and intact antibody each targets a different epitope (antigenic determinant) of the antigen; (ii) performing antigen/antibody reaction among the antibody fragment, the intact antibody, and the antigen; and (iii) assaying the mixture by FCS.
  • an antigenic protein e.g., a pathogenic protein such as an abnormal prion or a harmful protein such as an allergen protein
  • the fluorescence-labeled antibody fragment and the non-fluorescence-labeled intact antibody may form a complex via the antigen ( FIG. 1 ).
  • the present invention may relate to: (1) a method of quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy, comprising the use of a fluorescence-labeled antibody fragment targeted to an epitope of an antigen and a non-fluorescence-labeled intact antibody targeted to another epitope of the antigen to form an antigen/antibody complex among the antigen, the fluorescence-labeled antibody fragment, and the non-fluorescence-labeled intact antibody, and detecting and analyzing the formed antigen/antibody complex by fluorescence correlation spectroscopy; (2) the method of quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy according to (1), wherein the antigen is an antigenic protein; (3) the method of quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy according to (1) or (2), wherein the detection and analysis of the formed antigen/antibody complex by fluorescence correlation spectroscopy are a detection and an analysis of an antigen/
  • the present invention may also relate to: (6) a method of quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy, comprising the addition of a fluorescence-labeled antibody fragment targeted to an epitope of an antigen and a non-fluorescence-labeled intact antibody targeted to another epitope of the antigen to a test sample, performing antigen/antibody reaction thereamong, and detecting and analyzing an antigen/antibody complex formed among the antigen, the fluorescence-labeled antibody fragment, and the non-fluorescence-labeled intact antibody by fluorescence correlation spectroscopy; (7) the method of quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy according to (6), wherein the antigen is an antigenic protein; (8) the method of quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy according to (6) or (7), wherein the detection and/or assay of an antigen by fluorescence correlation spectroscopy
  • the present invention may further relate to: (11) the method of quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy according to (10), wherein the pathogenic protein antigen is an abnormal prion; (12) the method of quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy according to any one of (6) to (9), wherein the test sample is a food material, and the antigen is a harmful protein antigen contained in the food material; (13) a detection reagent for quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy, comprising a fluorescence-labeled antibody fragment targeted to an epitope of an antigen to be detected and/or assayed and a non-fluorescence-labeled intact antibody targeted to another epitope of an antigen to be detected and/or assayed; and (14) a kit for quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy, comprising the detection reagent according to
  • FIG. 1 is a diagram showing the outline of a method of quickly detecting and/or assaying an antigen-by FCS of the present invention.
  • FIG. 2 is a diagram showing the outline of a test on a difference in diffusion rate between a fluorescence-labeled antibody fragment alone and a complex of the fluorescence-labeled antibody fragment and a intact antibody in order to demonstrate the function of the method of quickly detecting and/or assaying an antigen by FCS of the present invention.
  • FIG. 3 is a diagram showing a result of the test on a difference in diffusion rate between a fluorescence-labeled antibody fragment alone and a complex of the fluorescence-labeled antibody fragment and a intact antibody in order to demonstrate the function of the method of quickly detecting and/or assaying an antigen by FCS of the present invention.
  • FIG. 4 is a diagram showing the outline of an apparatus for FCS assay used in the present invention.
  • FIG. 5 is a diagram showing the outline of preparation of a fluorescence-labeled antibody fragment used in the method of quickly detecting and/or assaying an antigen by FCS of the present invention.
  • FIG. 6 is a diagram showing a result of using a fluorescence-labeled antibody fragment and a intact antibody to test a difference in diffusion time depending on the formation of a complex of the antibody and an antigenic protein in the Examples of the present invention.
  • a method of quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy (FCS) as disclosed in the present invention comprises using a fluorescence-labeled antibody fragment targeted to an epitope of the antigen and a non-fluorescence-labeled intact antibody targeted to another epitope of the antigen in order to form an antigen/antibody complex among the antigen, the fluorescence-labeled antibody fragment, and the non-fluorescence-labeled intact antibody, and detecting and analyzing the formed antigen/antibody complex by FCS.
  • the method of quickly detecting and/or assaying an antigen is performed by (i) adding a fluorescence-labeled antibody fragment targeted to an epitope of the antigen and a non-fluorescence-labeled intact antibody targeted to another epitope of the antigen to a test sample; (ii) performing antigen/antibody reaction by mixing the sample supplemented with the antibody; and (iii) detecting and analyzing an antigen/antibody complex formed among the antigen, the fluorescence-labeled antibody fragment, and the non-fluorescence-labeled intact antibody by FCS to thereby quickly detect and/or assay the presence, size, concentration, or the like of the antigen in the sample.
  • a treatment procedure in the detection and/or assay by FCS of the present invention is performed by a procedure of merely adding and mixing the sample to a detection reagent comprising the fluorescence-labeled antibody fragment and the non-fluorescence-labeled intact antibody, without undergoing a step of physically separating the antigen contained in the test sample. Therefore, the step of detecting and assaying the antigen can be performed automatically or semi-automatically.
  • antibodies specifically binding to the antigen are prepared.
  • the antibodies specifically binding to an antigen that are used in the present invention can include polyclonal antibodies and monoclonal antibodies, preferably the monoclonal antibodies in terms of their specificity, and preferably IgG class monoclonal antibodies.
  • the antigen to be detected is first purified and obtained.
  • the antigen can be prepared by isolation and purification from a donor source using purification means known in the art.
  • the antigenic protein can be obtained by a genetic engineering approach whereby microorganisms, animal cells, or the like are used and allowed to produce the antigenic protein, followed by purification.
  • the antigenic protein can be prepared, when possible, by a chemical peptide synthesis method.
  • the chemical peptide synthesis can adopt synthesis means known in the art. Examples thereof include azide, acid chloride, acid anhydride, mixed anhydride, DCC, active ester, carboimidazole, and oxidation-reduction methods.
  • mice or plants are sensitized to the antigen by use of a routine protocol to prepare the antibodies.
  • Any method such as a hybridoma method (Nature 256, 495-497, 1975), trioma method, human B cell hybridoma method (Immunology Today 4, 72, 1983), and EBV-hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985), which provide antibodies produced by cultures of continuous cell lines, can be used, for example, in monoclonal antibody preparation.
  • an antigen such as an antigenic protein
  • mammals such as rat, mice, or rabbits are sensitized by administering the antigenic protein as an antigen to them.
  • An adjuvant such as a Freund's complete adjuvant (FCA) or a Freund's incomplete adjuvant (FIA) can be used, if necessary.
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • the immunization is performed mainly by intravenous, hypodermic, or intraperitoneal injection.
  • a time interval between immunizations is not particularly limited, and 1 to 10 immunizations are performed at several-day to several-week intervals. One to sixty days after the final immunization day, antibody-producing cells are collected.
  • the antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells.
  • cell fusion is performed between the antibody-producing cells and myeloma cells.
  • Generally obtainable cell lines can be used as the myeloma cells to be fused with the antibody-producing cells.
  • the cell lines used have drug selectivity and possess the property of being inviable in the unfused form in a HAT selective medium (which contains hypoxanthine, aminopterin, and thymidine) but viable therein only in the form fused with the antibody-producing cells.
  • Hybridomas of interest are selected from the cells after the cell fusion treatment.
  • a usual cell culture method or ascites formation method can be adopted as a method of collecting monoclonal antibodies from the established hybridomas.
  • the antibodies can be purified by appropriately selecting methods known in the art such as ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and affinity chromatography, or combining these methods.
  • commercially available already-prepared antibodies if any, can be used as an antibody against the antigenic protein used in the present invention.
  • a fluorescence-labeled antibody fragment prepared from the antigen is used as a detection reagent for performing antigen/antibody reaction with the antigen and detecting the antigen.
  • an antibody that binds to an epitope of the antigen different from that for the non-fluorescence-labeled intact antibody used in the present invention is selected as an antibody used in the preparation of the fluorescence-labeled antibody fragment.
  • a intact antibody against the antigen is fragmented with an enzyme such as pepsin and papain and converted to a monomer by reduction with, for example, 2-mercaptomethylamine or 2-mercaptoethanol, followed by labeling to thereby prepare the fluorescence-labeled antibody fragment.
  • an enzyme such as pepsin and papain
  • a fluorescent dye is used in the labeling.
  • a fluorescent dye such as fluorescein isothiocyanate (FITC) and Alexa 532 is used.
  • FCS fluorescence correlation spectroscopy
  • FCS Fibrosilicates
  • a detection system using FCS can avoid a complicated Bound/Free separation step necessary for conventional biomolecule detection systems (e.g., ELISA) performed predominantly.
  • ELISA ELISA
  • large amounts of samples can be assayed automatically with high sensitivity in a short time.
  • FCS techniques are known, and any method can be used in the present invention unless it hinders the detection and assay of an object to be detected and assayed by the present invention (Protein, Nucleic Acid and Enzyme, Vol. 44, No. 9, 1431-1438, 1999; Bio Industry, April issue, p. 52-59, 2003; Japanese Laid-Open Patent Application No. 2001-272404; and Japanese Patent No. 3517241).
  • FIG. 4(A) shows a schematic view of an FCS (fluorescence correlation spectroscopy) apparatus.
  • Excitation light from a laser is erected to a sample solution on a cover glass via a dichroic mirror (DM) and an objective lens.
  • Emitted fluorescence passes through a long pass or band pass filter (F) and is directed to an avalanche photodiode detector (APD) or a photomultiplier (PMT) after the background light outside the confocal surface is removed by a confocal pinhole.
  • APD avalanche photodiode detector
  • PMT photomultiplier
  • FIG. 4(B) shows a magnified schematic view of the observation region. It shows the state where a fluorescent molecule moving in Brownian motion passes through the confocal region narrowed down to the limit by the objective lens.
  • FIG. 4(C) shows a correlation curve after fluorescence correlation analysis.
  • the physical parameters of molecules such as “number” or “size” are obtained by analyzing the observed fluctuation of fluorescence intensity by use of formulas.
  • fluorescence from the exceedingly small region (approximately 400 nm in diameter, approximately 2 ⁇ m in axis length, less than 10 ⁇ 16 l in volume) of the sample solution is detected by using a confocal optical system ( FIG. 4 ).
  • MF20 manufactured by Olympus Corp. was used as an apparatus for FCS assay.
  • the assay was practiced by a scheme wherein three 30-second measurements were performed at wavelengths of 543 and 633 nm.
  • the observed fluctuation of fluorescence intensity is analyzed by use of formulas (1) to (4) described below to thereby obtain the physical parameters of molecules such as “number” or “size.”
  • an autocorrelation function is used to draw information from the signal of fluctuation.
  • the autocorrelation function used in FCS is represented by the formula (1).
  • s z/w, a ratio of the semimajor axis (z) of the observation region to the radius (w) thereof;
  • ⁇ D is called diffusion time (or correlation time) and represents average time when the fluorescent molecules pass through the observation region by diffusion; and N represents the average number of the molecules present in the observation region within a fixed time.
  • the analysis of the fluctuation by the formula (1) gives a curve as shown in FIG. 4 (C), from which the diffusion time ⁇ D showing the “mobility” of the molecules and the “number” N of the molecules are obtained.
  • the correlation curve shifts to the right with increases in molecular size, for example, as a result of association of the fluorescent molecules with other molecules, whereas it shifts to the left with decreases in molecular size, for example, as a result of dissociation.
  • the diffusion time ⁇ D obtained by the formula (1) stands in the relationship represented by the formula (2) with the diffusion constant D.
  • ⁇ D w 2 /4 D Formula 2
  • the diffusion constant D stands in the relationship represented by the formula (3) with the radius r of the molecule according to the Einstein-Stokes equation based on the assumption that molecules are spherical in shape.
  • ⁇ B , T, and ⁇ represent the Boltzmann constant, the absolute temperature, and the viscosity of a solvent, respectively.
  • the diffusion time ⁇ D can be linked as shown in the formula (4) to the radius r of the molecule corresponding to the “size” of the molecule according to the formulas (2) and (3).
  • the “number” and “size” of the molecules present in the observation region can be obtained by analyzing the fluctuation of fluorescence intensity by use of the formulas (1) and (4).
  • Detailed explanation about the fluctuation and the autocorrelation function are described in texts (T. Musha, “World of Fluctuation” Kodansha Bluebacks series, Kodansha Ltd., 1980; D. Eisenberg, et al., “Physical Chemistry With Applications To The Life Sciences”, the second volume, Baifukan Co., Ltd., p. 596-600, 1988; and M. Hino, “Spectrum Analysis”, Asakura Publishing Co., Ltd., p. 25-39, 1977).
  • Procedures in the detection and/or assay of an antigenic protein by FCS of the present invention and time required therefor are indicated in comparison to those in a method of detecting and/or assaying an antigenic protein by using a conventional ELISA method.
  • the present invention describes a method of detecting and/or assaying an antigen by fluorescence correlation spectroscopy (FCS) that can detect and assay an antigen independently of the shape or molecular weight of the antigen.
  • FCS fluorescence correlation spectroscopy
  • This method is applicable to antigens over a wide scope, including those with a relatively small molecular weight such as an antigenic protein (a pathogenic protein such as an abnormal prion, or a harmful protein) contained in a food material.
  • the detection and/or assay method of the present invention performs the detection and/or assay by FCS.
  • a treatment procedure for the detection and assay by FCS is performed by a procedure of merely mixing a test sample (which contains an antigen) to a detection reagent, comprised of a fluorescence-labeled antibody fragment and a non-fluorescence-labeled intact antibody, and performing an antigen/antibody reaction.
  • a detection reagent comprised of a fluorescence-labeled antibody fragment and a non-fluorescence-labeled intact antibody, and performing an antigen/antibody reaction.
  • the assay results can be monitored in almost real time. Therefore, the method of the present invention is suitable for automatically or semi-automatically performing procedures from the mixing and reaction of the detection reagent with the test sample to the indication of the assay results.
  • the method of the present invention comprises a smaller number of steps associated with the analysis procedure than that in methods such as ELISA and Western blotting conventionally used in the assay of an antigenic protein such as a prion, and can assay several microliters to several dozen microliters of a sample. Therefore, it can economically assay large amounts of test samples.
  • the method of the present invention can be expected to be utilized as practical assay means of an antigenic protein.
  • a solution of 10 nM Fab 647 (mouse IgG antibody) alone and a Fab 647 mixture obtained by mixing Fab 647 with 100 nM intact antibody were applied to a 384-well plate (Olympus) blocked with N101 (NOF Corp.), followed by assay with MF20 (Olympus).
  • the assay was performed by 30 sec. ⁇ three measurements at a laser power set to 100 ⁇ W.
  • the processing software in MF20 was used to derive each parameter including diffusion time.
  • the diffusion time of the labeled antibody fragment alone (Ab( ⁇ )) and the diffusion time of the complex (Ab(+), the fragment and the intact antibody) are, theoretically, approximately 600 ⁇ s and 900 ⁇ s, respectively. Their measurement values were also approximately 600 ⁇ s and 950 ⁇ s, respectively, indicating a significant difference in diffusion rate between the labeled antibody fragment alone and the labeled antibody fragment/antibody complex.
  • the fluorescence-labeled antibody fragment (Fab′-Alexa 532) of an anti-prion antibody was taken as an example.
  • the outline of preparation of Fab′-Alexa 532 is shown in FIG. 5 .
  • An anti-PrP antibody solution was equilibrated with a citric acid solution (pH 6.3) by use of a PD-10 column (Pharmacia) and then supplemented (37° C., approximately 30 minutes) with pepsin (1% (w/w)) to prepare F(ab′) 2 .
  • the degree of the digestion was confirmed by HPLC (column: G300SWXL).
  • the fragment was purified by FPLC (column: Superdex 200 (16/60)) using 0.1 M phosphate buffer solution (pH6.3), then concentrated, and stored. It was further reduced (37° C., approximately 1.5 hours) by the addition of 2-mercaptomethylamine (0.01 M) to prepare Fab′.
  • the Fab′-Alexa 532 fluorescence-labeled antibody fragment
  • the prion protein antigenic protein
  • the intact antibody anti-bovine recombinant prion antibody
  • 8.76E-7M was added in this order to a 384-well plate (Olympus Corp.) blocked with N101 (NOF Corp.) and well mixed with a pipette.
  • the plate was left at 37° C. for 1 hour, followed by assay with MF20 (apparatus for FCS assay; Olympus Corp).
  • the assay was performed by three 30-second measurements at a laser power set to 150 ⁇ W.
  • the processing software in MF20 was used to derive each parameter including diffusion time.
  • the molecular weight of the prion protein is approximately 30 kDa, and therefore, a significant difference in diffusion time might not arise if the intact antibody is not used.
  • the theoretical diffusion times of the fluorescence-labeled antibody fragment and a complex (fluorescence-labeled antibody fragment+prion protein) are approximately 600 ⁇ s and 650 ⁇ s, respectively.
  • the diffusion times of the fluorescence-labeled antibody fragment and the complex (fluorescence-labeled antibody fragment+prion protein) were approximately 600 ⁇ s and 650 ⁇ s, respectively, indicating no significant difference ( FIG. 6 ).
  • the theoretical diffusion times of the fluorescence-labeled antibody fragment and a complex are approximately 600 ⁇ s and 900 ⁇ s, respectively, and are significantly different.
  • the diffusion times of the fluorescence-labeled antibody fragment and the complex were approximately 600 ⁇ s and 950 ⁇ s, respectively, close to the theoretical values, indicating a significant difference ( FIG. 6 ).
  • this Example demonstrated that an antigen with a small molecular weight that cannot be detected unless the method of the present invention is used can be detected by using the present method.
  • a method of quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy comprising: (i) using a fluorescence-labeled antibody fragment targeted to an epitope of the antigen and a non-fluorescence-labeled intact antibody targeted to another epitope of the antigen in order to form an antigen/antibody complex among the antigen, the fluorescence-labeled antibody fragment, and the non-fluorescence-labeled intact antibody, and (ii) detecting and analyzing the formed antigen/antibody complex by fluorescence correlation spectroscopy.
  • a method of quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy comprising (i) adding a fluorescence-labeled antibody fragment targeted to an epitope of an antigen and a non-fluorescence-labeled intact antibody targeted to another epitope of an antigen to a test sample, (ii) performing antigen/antibody reaction there among, and (iii) detecting and analyzing an antigen/antibody complex formed among the antigen, the fluorescence-labeled antibody fragment, and the non-fluorescence-labeled intact antibody by fluorescence correlation spectroscopy.
  • test sample is a biological protein sample
  • antigen is a pathogenic protein antigen
  • test sample is a food material
  • antigen is a harmful protein antigen contained in the food material
  • a detection reagent for quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy comprising a fluorescence-labeled antibody fragment targeted to an epitope of an antigen to be detected and/or assayed and a non-fluorescence-labeled intact antibody targeted to another epitope of an antigen to be detected and/or assayed.
  • a kit for quickly detecting and/or assaying an antigen by fluorescence correlation spectroscopy comprising a detection reagent according to paragraph 12 or 13.

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US9040305B2 (en) 2004-09-28 2015-05-26 Singulex, Inc. Method of analysis for determining a specific protein in blood samples using fluorescence spectrometry
US9239284B2 (en) 2007-12-19 2016-01-19 Singulex, Inc. Scanning analyzer for single molecule detection and methods of use

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